Diagnosing glomerular diseases accurately and in timely fashion is key to developing a successful treatment plan and thereby prevent their progression to ESRD (end stage renal disease). However, their successful diagnosis remains challenging due to the complex diagnostic procedures used including invasive renal biopsies. Thus, there is a compelling need to develop simplified procedures to detect glomerular disease patients with high accuracy and efficiency. The primary goal of this STTR proposal is to develop a novel cell-based assay that will serve as a non-invasive diagnostic clinical tool to detect a form of glomerular disease commonly known as recurrent focal and segmental glomerulosclerosis (rFSGS). Importantly, this revolutionary concept can be extended to develop similar assays for other glomerular diseases and therefore, reduce or eliminate the need for renal biopsies. This will be the long-term goal of our company InDepth pharmaceuticals. Although there are multiple etiologies for FSGS, dysfunction of podocytes leading to cell death and proteinuria are primary outcomes of all forms of FSGS. This Phase I study involves development of an assay that will specifically diagnose rFSGS patients in which, FSGS recurs following renal transplant within hours to weeks and affects more than a third of FSGS patients. Thus, timely diagnosis of rFSGS patients will prevent ineffective renal transplants that are destined to fail. Since FSGS targets podocyte damage and death, our unique approach involved mRNA profiling of cultured podocytes treated with rFSGS patient plasma to reveal upregulated genes involved in cellular damage. Based on maximal upregulation from the profiling data, we selected three proapoptotic candidate rFSGS responsive genes IL1-?, BMF, and IGFBP3 that were specifically elevated in rFSGS patient plasma treated podocytes, their promoter regions were identified and cloned into a luciferase-based reporter vector and transfected into podocytes/HEK293 to generate stable cell lines. Strikingly, when these cell lines were exposed to plasma from rFSGS patients, increased reporter activity was noted; in contrast, no reporter activity was noted with all the other glomerular disease patients tested. Remarkably, the statistical analysis showed more than 80% specificity in detecting rFSGS patients. To further test our concept and enhance the specificity and robustness of our assay; in the Specific Aim 1, using the identified gene promoters we will construct cell lines that show lower variability and enhanced responsiveness to rFSGS plasma, by employing two parallel approaches, including a CRSPR/Cas system for directed insertion of promoter-reporter construct at the Safe Harbor site AAVS1 (locus PPP1R12C) in cellular genome and an alternate approach, that involves conventional transfection followed by clonal selection to identify clone/s with highest magnitude of induction. In the Specific Aim 2 we will conduct initial validation of constructed cell lines by measuring their responses towards plasma from various nephropathy patients. Several rFSGS and non-rFSGS patient plasma are available through various indigenous and collaborative efforts between MUSC and InDepth pharmaceuticals to power this study.